天津医药

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靶向人乳头瘤病毒18E6的短发夹结构RNA慢病毒载体的构建

罗远材1,瞿全新2,糜若然3,4   

  1. 1. 天津市第一中心医院
    2. 天津第一中心医院
    3.
    4. 天津医科大学总医院妇产科
  • 收稿日期:2011-01-21 修回日期:2011-03-31 出版日期:2011-09-15 发布日期:2011-09-15
  • 通讯作者: 罗远材

Construction of a shRNA Lentiviral Vector Aiming at The Target of HPV18E6 Oncogene

  • Received:2011-01-21 Revised:2011-03-31 Published:2011-09-15 Online:2011-09-15

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摘要： 摘要 目的：构建含有靶向人乳头瘤状病毒18亚型E6癌基因（HPV18E6）的短发夹结构RNA（shRNA）的慢病毒载体。方法：根据HPV18E6癌基因的相关信息，设计并合成shRNA干扰序列，连入pGCSIL-GFP质粒，构建含有shRNA的重组质粒pGC-shRNA并鉴定；将重组质粒pGC-shRNA和病毒包装质粒pHelper 1.0、pHelper 2.0共转染293T 细胞，进行病毒包装，收集病毒液并进行浓缩、纯化；所得病毒液感染293T细胞，测定病毒滴度。结果：经同源性检索及对转录后得到的RNA进行二级结构预测，HPV18E6的shRNA干扰序列与人类基因组没有同源性，此插入序列转录后得到的RNA二级结构能形成发夹状结构；重组质粒pGC-shRNA经PCR鉴定及DNA测序结果显示该重组质粒构建成功；成功对病毒进行包装、浓缩及纯化后，所得病毒滴度为3x108TU/ml。结论：成功构建了HPV18E6-RNAi-LV慢病毒载体，为后续RNA干扰实验的开展提供了有力的工具。

关键词: HPV18E6, 短发夹结构RNA, 重组质粒, 慢病毒载体, RNA干扰

Abstract: Abstract Objective：To construct a lentiviral vector which contains short hairpin RNA(shRNA) interfering sequence aiming at the target of human Papillomavirus 18E6 (HPV18E6) oncogene.Method：Based on the information of HPV18E6 oncogene,design and synthesize shRNA interfering sequence,recombinate into pGCSIL-GFP plasmid to construct a recombination plasmid pGC-shRNA which contains shRNA interfering sequence and apprase it；Recombination plasmid and packaging plasmid pHelper 1.0 and pHelper 2.0 co-transduce the 293T cell to package the lentiviral vector,collects the lentiviral vector solution,concentrates and purifies it；The lentiviral vector solution after concentrating and purifying transduce 293T cell to determine the concentration of the lentiviral vector solution.Results：Indexing shRNA of interfering sequence aiming at the target of HPV18E6 oncogene with human genes in BLASTER,there is no homogeneous,the second constructure of the transcripts of interfering sequence can conform short hairpin；The recombination plasmid pGC-shRNA was obtained with the correct sequence in the insert after the appraisal；After packaging and collecting and concentrating the lentiviral vector successfully, the concentration of lentiviral vector solution is 3x108TU/ml.Conclusion：The lentiviral vectors of HPV18E6-RNAi-LV was constructed successfully,which provides practical tool for later study.

Key words: HPV18E6, short hairpin RNA, recombination plasmid, lentiviral vector, RNA interfere